High-resolution mass spectrum characterization method of chromatographic co-outflow dimethylphenol isomeride and application of high-resolution mass spectrum characterization method

Abstract

The invention discloses a high-resolution mass spectrometry characterization method for chromatography co-outflow dimethylphenol isomeride and application, and the method comprises the following steps: selecting a corresponding sample pretreatment process to prepare a sample according to the petroleum content range in the sample; the method comprises the following steps: in a methanol-5. 0-6.5 mmol / L ammonia water mobile phase system, detecting a standard sequence sample, a conversion factor calibration sample and the sample by utilizing LC-HRMS (Liquid Chromatography-High Resolution Mass Spectrometry), and collecting primary mass spectrum full scanning (MS1) and selective ion secondary mass spectrum scanning (MS2) data; according to the MS1 data, calculating a first standardized concentration of the 3, 5-dimethylphenol and the 2, 6-dimethylphenol which flow out together, and a first conversion factor of the 2, 6-dimethylphenol to the 3, 5-dimethylphenol; the concentration of other 11 phenols and alkyl substituted phenols; and according to the MS2 data, calculating a second standardized concentration and a second conversion factor of the co-effluent component, and further calculating the concentrations of 3, 5-dimethylphenol and 2, 6-dimethylphenol. According to the method, the quantitative characterization of 13 C0-C2-alkyl substituted phenol single components is realized.

Description

Technical Field

[0001] The invention relates to the technical field of environmental detection, and in particular to a high-resolution mass spectrometry characterization method and application of chromatographic co-eluting dimethylphenol isomers. Background Art

[0002] The stripping water discharged from the sulfur-containing and salt-containing wastewater stripping device is oily wastewater and is the main process wastewater in oil refineries. The unit discharge volume reaches 0.2m 3 / t crude oil, the volatile phenol content is as high as 0.2-0.5kg / m 3 (Yan Song, Li Lingbo, Han Congbi, Zhou Yanhong. Study on organic components of high-sulfur and high-acid crude oil wastewater [J]. Contemporary Chemical Industry, 2015, 44(09): 2081-2083). Coal gasification is an important process in modern coal chemical projects, among which Lurgi pressurized gasification is a relatively mature gasification process. The scrubbing wastewater produced by gasification contains high-concentration difficult-to-degrade organic pollutants such as phenol and ammonia (Li Xueyuan, Wei Danqing. Detection of phenolic compounds in coal gasification wastewater by gas chromatography-mass spectrometry [J]. Modern Chemical Industry, 2015, 35(09): 187-188). The high content of volatile phenolic substances in stripping water and coal gasification wastewater mainly includes phenol, methylphenol (3 isomers) and dimethyl / ethylphenol (9 isomers), which have recovery and reuse value. Clarifying the composition of volatile phenolic substances is the basis for the recovery and comprehensive utilization of volatile phenolic substances.

[0003] Volatile phenol components contain many isomers. When using chromatography-mass spectrometry for quantification, it is necessary to achieve chromatographic separation of isomers, and chromatographic separation of isomers has always been a technical difficulty in this field. Liu Yulong et al. found that the elution order and chromatographic co-elution of dimethylphenol isomers in the same reverse C18 liquid chromatography column changed under two different mobile phase systems, and established a method for calculating the single component content of chromatographic co-elution isomers, realizing the quantitative characterization of the above 13 alkyl-substituted phenol single components in oily wastewater (Liu Yulong, Wang Zixian, Zhang Xiaofei, Shao Zhiguo, Tong Kun. A characterization method and application of C0~C2-alkyl-substituted phenols. Chinese patent: 202211482228.X; Liu Yulong, Shao Zhiguo, Tong Kun, Zhang Xiaofei, Wang Zixian. A detection method and application of C1~C2-alkyl-substituted phenol isomers. Chinese patent: 202211482206.3). In the prior art, whether using gas chromatography separation or liquid chromatography separation technology, it is rare to solve the problem of accurate quantification of a single component of chromatographic co-eluting isomers under only one mobile phase system. Summary of the invention

[0004] The present invention aims to provide a high-resolution mass spectrometry characterization method and application of chromatographic co-eluting dimethylphenol isomers, which adopts liquid chromatography-high-resolution mass spectrometry (LC-HRMS) coupling technology, and simultaneously collects a primary mass spectrometry full scan (MS) in a methanol-ammonia mobile phase system. 1 ) and selected ion secondary mass spectrometry (MS 2 ) data to achieve quantitative characterization of 13 C0-C2-alkyl substituted phenol single components in oily wastewater. In addition, the present invention is also applicable to the quantitative characterization of 13 C0-C2-alkyl substituted phenol single components in drinking water, surface water and groundwater, coal gasification wastewater, and oily solid waste.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] In one aspect, the present invention provides a high-resolution mass spectrometry characterization method for chromatographically co-eluting dimethylphenol isomers, the method comprising the following steps:

[0007] According to the petroleum content range in the sample, select the corresponding sample pretreatment process to prepare the sample;

[0008] In the mobile phase system of methanol-5.0~6.5mmol / L ammonia water, the standard sequence samples, the conversion factor calibration samples and the sample were detected by LC-HRMS, and the primary mass spectrometer full scan (MS 1 ) and selected ion secondary mass spectrometry (MS 2 )data;

[0009] According to MS 1 data, calculate the first standardized concentrations of coeluting 3,5-dimethylphenol and 2,6-dimethylphenol, and the first conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol by internal standard calibration; and the concentrations of phenol, 2-methylphenol, 3-methylphenol, 4-methylphenol, 2,3-dimethylphenol, 2,4-dimethylphenol, 2,5-dimethylphenol, 3,4-dimethylphenol, 2-ethylphenol, 3-ethylphenol, and 4-ethylphenol;

[0010] According to MS 2 The data were used to calculate the second standardized concentrations of co-eluted 3,5-dimethylphenol and 2,6-dimethylphenol, and the second conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol by internal standard calibration method;

[0011] The concentrations of 3,5-dimethylphenol and 2,6-dimethylphenol are calculated based on the first standardized concentration and the second standardized concentration of 3,5-dimethylphenol and 2,6-dimethylphenol; and the first conversion factor and the second conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol.

[0012] As a preferred embodiment, the sample preparation process is selected according to the petroleum content range in the sample, specifically including:

[0013] When the petroleum content in the sample does not exceed 50 mg / L, the sample is filtered through a 0.22 μm filter membrane before sampling and testing;

[0014] When the petroleum content in the sample exceeds 50 mg / L, the sample is alkalized with NaOH and extracted with dichloromethane and n-hexane, and the organic phase is separated by centrifugation. The aqueous phase is filtered through a 0.22 μm filter membrane, and then neutralized with formic acid before sampling and testing.

[0015] As a preferred embodiment, the elution conditions of the methanol-5.0-6.5 mmol / L ammonia water mobile phase system include:

[0016] The column flow rate was 0.20 mL / min;

[0017] Gradient elution was as follows: maintaining zero gradient elution for 1 min, then isocratic elution to increase the volume ratio of methanol to 40% within 30 min, and maintaining the volume ratio of methanol at 40% until all components were eluted;

[0018] The column temperature is 35-40°C.

[0019] As a preferred embodiment, the elution conditions of the methanol-5.0-6.5 mmol / L ammonia water mobile phase system include:

[0020] The column flow rate was 0.20 mL / min;

[0021] Gradient elution: 0-1 min, 20% methanol; 1-31 min, 20%-40% methanol; 31-32 min, 40% methanol; 32-32.1 min, 40%-90% methanol; 32.1-37 min, 90% methanol; 37-37.1 min, 90%-20% methanol; 37.1-42 min, 20% methanol;

[0022] The column temperature was 40°C.

[0023] As a preferred embodiment, the selected ion secondary mass spectrometry (MS 2 ), the parent ion of 3,5-dimethylphenol-2,4,6-D3 is 124.08472 m / z, and the quantitative daughter ion is 96.05342 m / z; the parent ions of 3,5-dimethylphenol and 2,6-dimethylphenol are 121.06589 m / z, and the quantitative daughter ion is 93.03459 m / z.

[0024] As a preferred embodiment, the calculation formula of the first conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol is:

[0025]

[0026] Where k1 is the first conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol, A 3,5 / 2,6-DMP，1 is the quantitative precursor ion peak area of ​​the co-eluting components 3,5-dimethylphenol and 2,6-dimethylphenol in the calibration points of the calibration sample series, A 3,5-DMP，1 The quantitative parent ion peak area of ​​3,5-dimethylphenol in the calibration sample is A IS(3,5 / 2,6-DMP)，1 and A IS(3,5-DMP)，1 The peak areas of the corresponding internal standard quantitation precursor ions in the calibration samples are calculated for the calibration points and conversion factors of the calibration sample series.

[0027] As a preferred embodiment, the calculation formula of the second conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol is:

[0028]

[0029] Where k2 is the second conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol, A 3,5 / 2,6-DMP，2 is the quantitative product ion peak area of ​​the co-eluting components 3,5-dimethylphenol and 2,6-dimethylphenol at the calibration points of the calibration sample series, A 3,5-DMP，2 The quantitative product ion peak area of ​​3,5-dimethylphenol in the calibration sample is A IS(3,5 / 2,6-DMP)，2 and A IS(3,5-DMP)，2 The peak areas of the corresponding internal standard quantifier ions in the calibration samples are calculated for the calibration points and conversion factors for the calibration sample series.

[0030] As a preferred embodiment, the concentration of 3,5-dimethylphenol and 2,6-dimethylphenol is calculated as follows:

[0031]

[0032]

[0033] In the formula, c 2,6-DMP represents the concentration of 2,6-dimethylphenol, c 3,5-DMP represents the concentration of 3,5-dimethylphenol, c STD1 represents the first normalized concentration of coeluting 3,5-dimethylphenol and 2,6-dimethylphenol, c STD2represents the second standardized concentration of co-eluted 3,5-dimethylphenol and 2,6-dimethylphenol, k1 represents the first conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol, and k2 represents the second conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol.

[0034] On the other hand, the present invention provides an application of a high-resolution mass spectrometry characterization method for chromatographic co-eluting dimethylphenol isomers, which is applied to the quantitative characterization of 13 C0~C2-alkyl substituted phenol single components in oily wastewater, coal gasification wastewater, oily solid waste, drinking water, surface water and groundwater.

[0035] Technical effects and advantages of the present invention:

[0036] (1) Use a single injection and collect a full mass spectrometer scan (MS 1 ) and selected ion secondary mass spectrometry (MS 2 ) data, and achieved accurate quantification of the single components of chromatographically co-eluted 3,5-dimethylphenol and 2,6-dimethylphenol isomers.

[0037] (2) Based on the parent ion 121.06589 m / z and characteristic daughter ion 93.03459 m / z of 3,5-dimethylphenol and 2,6-dimethylphenol, a single-component quantitative method for chromatographic co-elution isomers was established by solving a set of equations.

[0038] (3) The sample is not enriched or derivatized, and the detection limit of the method for 13 C0-C2-alkyl-substituted phenols is less than 1.0 μg / L. Compared with the standard HJ744-2015 (derivation-GC-MS method) for testing phenolic compounds, the sensitivity of this method is 1 to 2 orders of magnitude higher.

[0039] Other features and advantages of the present invention will be described in the following description, and partly become obvious from the description, or be understood by implementing the present invention. The purpose and other advantages of the present invention can be realized and obtained by the structures pointed out in the description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The present invention is a flow chart of a high-resolution mass spectrometry characterization method for chromatographically co-eluting dimethylphenol isomers;

[0041] Figure 2 The present invention is a method for analyzing 9 C2-alkyl substituted phenols [MH] in a methanol-6.0 mmol / L ammonia mobile phase system. - Extracted ion map of

[0042] Figure 3It is the secondary mass spectrum of 3,5-dimethylphenol parent ion 121.06589 m / z under normalized collision energy (NCE=60, 80, 100).

[0043] Figure 4 It is the secondary mass spectrum of 2,6-dimethylphenol parent ion 121.06589 m / z under normalized collision energy (NCE=60, 80, 100). DETAILED DESCRIPTION

[0044] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] In order to solve the deficiencies of the prior art, the present invention discloses a high-resolution mass spectrometry characterization method for chromatographically co-eluted dimethylphenol isomers. Figure 1 The flowchart of the high-resolution mass spectrometry characterization method of the chromatographic co-eluting dimethylphenol isomers of the present invention is as follows: Figure 1 As shown, the method comprises the following steps:

[0046] Step S1, according to the petroleum content range in the sample, select the corresponding sample pretreatment process to prepare the sample;

[0047] Step S2, using LC-HRMS to detect the standard sequence samples, the conversion factor calibration samples and the sample in a methanol-5.0-6.5 mmol / L ammonia water mobile phase system, collecting a primary mass spectrometer full scan (MS 1 ) and selected ion secondary mass spectrometry (MS 2 )data;

[0048] Step S3: According to MS 1 data, calculate the first standardized concentrations of coeluting 3,5-dimethylphenol and 2,6-dimethylphenol, and the first conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol by internal standard calibration; and the concentrations of phenol, 2-methylphenol, 3-methylphenol, 4-methylphenol, 2,3-dimethylphenol, 2,4-dimethylphenol, 2,5-dimethylphenol, 3,4-dimethylphenol, 2-ethylphenol, 3-ethylphenol, and 4-ethylphenol;

[0049] Step S4: According to MS 2The data were used to calculate the second standardized concentrations of co-eluted 3,5-dimethylphenol and 2,6-dimethylphenol, and the second conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol by internal standard calibration method;

[0050] Step S5, calculating the concentrations of 3,5-dimethylphenol and 2,6-dimethylphenol according to the first standardized concentration and the second standardized concentration of 3,5-dimethylphenol and 2,6-dimethylphenol; and the first conversion factor and the second conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol.

[0051] In step S1 of the present invention, the sample is prepared by selecting a corresponding sample pretreatment process according to the petroleum content range in the sample, which specifically includes:

[0052] When the petroleum content in the sample does not exceed 50 mg / L, the sample is filtered through a 0.22 μm filter membrane before sampling and testing;

[0053] When the petroleum content in the sample exceeds 50 mg / L, the sample is alkalized with NaOH and extracted with dichloromethane and n-hexane, and the organic phase is separated by centrifugation. The aqueous phase is filtered through a 0.22 μm filter membrane, and then neutralized with formic acid before sampling and testing.

[0054] In step S2 of the present invention, the elution conditions of the methanol-5.0-6.5 mmol / L ammonia water mobile phase system include:

[0055] The column flow rate was 0.20 mL / min;

[0056] Gradient elution was as follows: maintaining zero gradient elution for 1 min, then isocratic elution to increase the volume ratio of methanol to 40% within 30 min, and maintaining the volume ratio of methanol at 40% until all components were eluted;

[0057] The column temperature is 35-40°C.

[0058] Preferably, the elution conditions of the methanol-5.0-6.5 mmol / L ammonia water mobile phase system include:

[0059] The column flow rate was 0.20 mL / min;

[0060] Gradient elution: 0-1 min, 20% methanol; 1-31 min, 20%-40% methanol; 31-32 min, 40% methanol; 32-32.1 min, 40%-90% methanol; 32.1-37 min, 90% methanol; 37-37.1 min, 90%-20% methanol; 37.1-42 min, 20% methanol;

[0061] The column temperature was 40°C.

[0062] In step S3 of the present invention, the selected ion secondary mass spectrometry (MS 2 ), the parent ion of 3,5-dimethylphenol-2,4,6-D3 is 124.08472 m / z, and the quantitative daughter ion is 96.05342 m / z; the parent ions of 3,5-dimethylphenol and 2,6-dimethylphenol are 121.06589 m / z, and the quantitative daughter ion is 93.03459 m / z.

[0063] In step S3 of the present invention, the calculation formula of the first conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol is:

[0064]

[0065] Where k1 is the first conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol, A 3,5 / 2,6-DMP，1 is the quantitative precursor ion peak area of ​​the co-eluting components 3,5-dimethylphenol and 2,6-dimethylphenol in the calibration points of the calibration sample series, A 3,5-DMP，1 The quantitative parent ion peak area of ​​3,5-dimethylphenol in the calibration sample is A IS(3,5 / 2,6-DMP)，1 and A IS(3,5-DMP)，1 The peak area of ​​the internal standard quantitation precursor ion in the calibration sample is calculated for the calibration points and conversion factors of the above calibration sample series.

[0066] In step S4 of the present invention, the calculation formula of the second conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol is:

[0067]

[0068] Where k2 is the second conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol, A 3,5 / 2,6-DMP，2 is the quantitative product ion peak area of ​​the co-eluting components 3,5-dimethylphenol and 2,6-dimethylphenol at the calibration points of the calibration sample series, A 3,5-DMP，2 The quantitative product ion peak area of ​​3,5-dimethylphenol in the calibration sample is A IS(3,5 / 2,6-DMP)，2 and A IS(3,5-DMP)，2 The peak area of ​​the corresponding internal standard quantifier ion in the calibration sample is calculated for the calibration points and conversion factors of the above calibration sample series.

[0069] In step S5 of the present invention, the concentration of 3,5-dimethylphenol is c 3,5-DMP and the concentration of 2,6-dimethylphenol c 2,6-DMP The calculation formula is:

[0070]

[0071]

[0072] In the formula, c 2,6-DMP represents the concentration of 2,6-dimethylphenol, c 3,5-DMP represents the concentration of 3,5-dimethylphenol, c STD1 represents the first normalized concentration of coeluting 3,5-dimethylphenol and 2,6-dimethylphenol, c STD2 represents the second standardized concentration of co-eluted 3,5-dimethylphenol and 2,6-dimethylphenol, k1 represents the first conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol, and k2 represents the second conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol.

[0073] Example 1

[0074] This example provides two sample pretreatment methods for LC-HRMS injection testing.

[0075] 1. Stripping water or coal gasification wastewater with petroleum content ≤50mg / L

[0076] The water sample was filtered through a 0.22 μm filter membrane. Depending on the volatile phenol content, 10 to 900 μL of the filtrate was taken to prepare the sample.

[0077] This process is also applicable to the sample preparation of relatively clean water samples such as drinking water, groundwater and surface water; or the sample preparation of oil-water mixtures such as oily wastewater.

[0078] 2. Stripping water or coal gasification wastewater with petroleum content > 50 mg / L

[0079] Take 10.00mL of water sample in a 20mL round-bottom screw-mouth sample bottle (with a sample cap of polytetrafluoroethylene liner), add 100μL of 2.5mol / L NaOH solution to adjust pH ≥ 12, add 1-3mL of dichloromethane-n-hexane (2+1) mixed solvent, and tighten the cap. Shake for 30 minutes and centrifuge. Take the aqueous phase and add formic acid to neutralize it, filter the aqueous phase through a 0.22μm filter membrane, and take 10-900μL of the filtrate to prepare the sample according to the volatile phenol content.

[0080] Example 2

[0081] In this example, the chromatographic conditions of 9 C2-alkyl substituted phenols were optimized under a methanol-ammonia mobile phase system, and the effect of ammonia mobile phase concentration in the range of 4.0 to 8.0 mmol / L on the chromatographic separation of C2-alkyl substituted phenols was investigated.

[0082] This example and the following examples all use ACQUITY UPLC BEH C 18A chromatographic column (1.7 μm, 2.1 mm×150 mm, Waters, USA) was used to separate C1-C2-alkyl substituted phenols.

[0083] Under the conditions of 0.20mL / min column flow rate and 6.0mmol / L ammonia mobile phase concentration, the initial volume ratio of methanol mobile phase was set to 10%, 20% and 25% respectively, and zero gradient elution was maintained for 1min. Then the volume ratio of methanol was increased to 40% within 30min by isocratic elution, and the volume ratio of methanol was maintained at 40% until all components flowed out. The results showed that when the initial volume ratio of methanol was 10%, 20% and 25%, all C2-alkyl substituted phenols were eluted within 32min; when the initial volume ratio of methanol mobile phase was 10%, the separation of the first eluted components was relatively poor, while the separation of the later eluted components was relatively good; when the initial volume ratio of methanol mobile phase was 25%, the separation of the first eluted components was relatively good, while the separation of the later eluted components was relatively poor, so the initial volume ratio of methanol was selected to be 20%.

[0084] The effects of column temperatures of 30℃, 35℃, 40℃ and 45℃ on the chromatographic separation of C2-alkyl substituted phenols were compared. The chromatographic separation of C2-alkyl substituted phenols deteriorated when the column temperatures were 30℃ and 45℃, and better chromatographic separation was obtained when the column temperatures were 35℃ and 40℃. When the column temperature was 40℃, the column pressure decreased by about 40 to 50 bar, so the preferred column temperature was 40℃.

[0085] The optimized chromatographic conditions are as follows: column flow rate is 0.20 mL / min; gradient elution is: 0-1 min, 20% methanol; 1-31 min, 20%-40% methanol; 31-32 min, 40% methanol; 32-32.1 min, 40%-90% methanol; 32.1-37 min, 90% methanol; 37-37.1 min, 90%-20% methanol; 37.1-42 min, 20% methanol; column temperature is 35-40°C; preferably 40°C.

[0086] Under the optimized chromatographic conditions, the effect of ammonia mobile phase concentrations of 4.0, 6.0 and 8.0 mmol / L on the chromatographic separation of C2-alkyl substituted phenols was investigated. When the ammonia concentration was 4.0 mmol / L, 7 peaks were separated from the 9 C2-alkyl substituted phenols; when the ammonia concentration was 6.0 mmol / L, 8 peaks were separated with better peak shape; when the ammonia concentration was 8.0 mmol / L, only 7 peaks were separated.

[0087] The retention time and elution order of C2-alkyl substituted phenols at an ammonia concentration of 6.0 mmol / L were confirmed by a single calibration method, and the co-elution between components was determined. Figure 2The present invention is a method for analyzing 9 C2-alkyl substituted phenols [MH] in a methanol-6.0 mmol / L ammonia mobile phase system. - The extracted ion map of , where the corresponding chromatographic peaks are: 1, 3,4-dimethylphenol; 2, 3-ethylphenol; 3, 3,5-dimethylphenol; 4, 2,6-dimethylphenol; 5, 2,3-dimethylphenol; 6, 4-ethylphenol; 7, 2,5-dimethylphenol; 8, 2-ethylphenol; 9, 2,4-dimethylphenol.

[0088] like Figure 2 As shown in the figure, when the ammonia concentration is 6.0mmol / L, only 3,5-dimethylphenol and 2,6-dimethylphenol co-elute. Further experiments determined that when the ammonia concentration is 5.0-6.5mmol / L, 9 C2-alkyl substituted phenols have 8 peaks with good peak shapes and only 3,5-dimethylphenol and 2,6-dimethylphenol co-elute. Based on this, the appropriate ammonia mobile phase concentration range is determined to be 5.0-6.5mmol / L, and the ammonia mobile phase concentration of 6.0mmol / L is selected for sample testing.

[0089] Example 3

[0090] This embodiment provides a method for detecting 13 C0-C2-alkyl substituted phenol single components in stripping water using ultra-performance liquid chromatography-orbitrap high-resolution mass spectrometry (UPLC-Orbitrap HRMS).

[0091] 1. Instrument conditions

[0092] UPLC-Orbitrap HRMS: Vanquish C-H10 ultra-high performance liquid chromatograph, Q-Exactive Plus electrostatic field orbitrap high-resolution mass spectrometer (ThermoFisher, USA).

[0093] (1) Liquid chromatography conditions

[0094] Mobile phase system: methanol-6.0mmol / L ammonia water. Column temperature: 40℃; Column flow rate: 0.20mL / min; Sample chamber temperature: 6℃; Injection volume: 2μL. Gradient elution: 0-1min, 20% methanol; 1-31min, 20%-40% methanol; 31-32min, 40% methanol; 32-32.1min, 40%-90% methanol; 32.1-37min, 90% methanol; 37-37.1min, 90%-20% methanol; 37.1-42min, 20% methanol.

[0095] (2) Orbitrap HRMS conditions

[0096] Heated electrospray ion source: negative ion mode; sheath gas flow rate: 35Arb; auxiliary gas flow rate: 10Arb; spray voltage 2.6kV; ion transfer tube temperature: 320°C; S-lens RF voltage: 50V; auxiliary gas heater temperature: 380°C.

[0097] Full scan mass spectrometry (MS 1 ) Acquisition parameters: Resolution: 70000; Maximum capacity of C-trap ion number (AGCTarget): 1×10 6 . C-trap maximum injection time (Maximum IT): 100ms; mass range: m / z90~155;

[0098] Selected ion secondary mass spectrometry (MS 2 ) acquisition parameters: selected ions were 121.06589 m / z and 124.08472 m / z; resolution: 17500; maximum capacity of C-trap ion number (AGC Target): 1×10 5 ; C-trap maximum injection time (Maximum IT): 100 ms; isolation window: 1 m / z; normalized collision energy (steppedNCE): 60, 80 and 100.

[0099] 2. Preparation of standard sequence samples and conversion factor calibration samples

[0100] Single standard stock solutions were prepared using standard samples of 13 phenols (phenol, methylphenol and dimethyl / ethylphenol) and internal standards (phenol-D5 and 3,5-dimethylphenol-2,4,6-D3) with a purity greater than 98%. A mixed working solution of 13 phenols, a 3,5-dimethylphenol working solution and a mixed internal standard working solution were prepared by stepwise dilution with methanol. The working solutions were then used to prepare standard sequence samples and conversion factor calibration samples.

[0101] Standard series samples: Add 900 μL of pure water to 6 2mL sample bottles respectively, add 13 kinds of phenol mixed working solutions and mixed internal standard working solutions in sequence with a micro syringe, and add methanol to make the total amount of methanol in the calibration standard solution 100 μL. Shake well and store at 4-6℃ for testing. The concentrations of the 13 phenol components in the standard series are 10, 100, 500, 1000, 2500 and 5000 μg / L respectively, and the concentration of each internal standard component is 200 μg / L.

[0102] Conversion factor calibration sample: Add 900 μL of pure water to a 2 mL sample bottle, use a micro syringe to add 3,5-dimethylphenol working solution and mixed internal standard working solution in sequence, and add methanol to make the total amount of methanol in the calibration standard solution 100 μL. Shake well and store at 4-6°C for testing. The concentration of 3,5-dimethylphenol in the conversion factor calibration sample is 1000 μg / L, and the concentration of each component of the internal standard is 200 μg / L.

[0103] 3. Sample collection and specimen preparation

[0104] The stripping water or coal gasification wastewater samples were collected in 40mL VOA sample bottles with polytetrafluoroethylene liners. When sampling, the water sample was required to fill the sample bottle and not overflow outside the bottle, and no protective agent was added. The sample was refrigerated at 4-6℃, and the sample preparation was completed within 48 hours.

[0105] Take out the refrigerated water sample, wait for the sample temperature to return to room temperature, and filter it through a 0.22μm nylon filter. Pre-add 90μL of methanol to a 2mL sample bottle. Depending on the volatile phenol content, take 10-900μL of the filtrate and add it to the sample bottle, add pure water to make the total volume of the sample 990μL, then add 10μL of the mixed internal standard working solution, shake well, and store at 4-6℃ for testing.

[0106] 4. Data collection and result calculation

[0107] Xcalibur 4.4 software was used to collect data, and TraceFinder 5.1 software was used for data analysis. The target compounds in the samples were qualitatively confirmed based on retention time matching, and the parent ion [MH] - The concentration of the target compound was calculated by the peak area of ​​the mixed internal standard or product ion. Table 1 shows the retention time, quantitative parent ion and quantitative product ion information of the mixed internal standard and 13 phenolic components.

[0108] The effects of different collision energies on the secondary mass spectrometry fingerprint characteristics of 3,5-dimethylphenol and 2,6-dimethylphenol were investigated in a methanol-6.0mmol / L ammonia mobile phase system. The results showed that when the collision energy increased from NCE=30 to NCE=100, the parent ions of 3,5-dimethylphenol and 2,6-dimethylphenol (121.06589 m / z) both produced 93.03459 m / z ([C6H5O] - )’s characteristic daughter ions, higher collision energy can obtain higher daughter ion abundance; the relative abundance of other daughter ions is lower. Figure 3 It is the secondary mass spectrum of 3,5-dimethylphenol parent ion 121.06589 m / z under normalized collision energy (NCE=60, 80, 100). Figure 4The secondary mass spectrum of 2,6-dimethylphenol parent ion 121.06589 m / z at normalized collision energy (NCE = 60, 80, 100) is shown in Figure 2. Figure 3 and Figure 4 As shown in the figure, 3,5-dimethylphenol and 2,6-dimethylphenol do not have their own characteristic daughter ions that can be used for quantification; the parent ions of 3,5-dimethylphenol and 2,6-dimethylphenol are 121.06589 m / z ([C8H9O] - ) fragmented to produce a relatively abundant daughter ion of 93.03459 m / z ([C6H5O] - ), which can be applied to quantification.

[0109] Table 1 Quantitative parent ions and daughter ions of internal standard and 13 phenolic components

[0110]

[0111]

[0112] About MS 1 The data were processed and the concentrations of phenol, 2-methylphenol, 3-methylphenol, 4-methylphenol, 2,3-dimethylphenol, 2,4-dimethylphenol, 2,5-dimethylphenol, 3,4-dimethylphenol, 2-ethylphenol, 3-ethylphenol and 4-ethylphenol were calculated by the internal standard method using the peak area of ​​the parent ion.

[0113] The single component concentration of co-eluted 3,5-dimethylphenol and 2,6-dimethylphenol is calculated as follows:

[0114] When the prepared concentrations of 3,5-dimethylphenol (3,5-DMP) and 2,6-dimethylphenol (2,6-DMP) are equal at each calibration point, a calibration curve is drawn with the sum of the concentrations of 3,5-dimethylphenol and 2,6-dimethylphenol as the independent variable and the sum of the response values ​​(such as peak area) of the parent ion 121.06589 m / z of 3,5-dimethylphenol and 2,6-dimethylphenol divided by the parent ion response value (such as peak area) of the associated internal standard as the dependent variable. The concentration value of the co-eluting component calculated by the calibration curve is defined as the first standardized concentration of the co-eluting 3,5-dimethylphenol and 2,6-dimethylphenol, and is expressed as c STD1 express.

[0115] The first conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol is represented by k1 and is calculated according to formula (1):

[0116]

[0117] Among them, A 3,5 / 2,6-DMP，1A is the quantitative parent ion peak area of ​​the co-eluting components 3,5-dimethylphenol and 2,6-dimethylphenol at the calibration point (e.g. 1000 μg / L) of the calibration sample series, 3,5-DMP，1 The quantitative parent ion peak area of ​​3,5-dimethylphenol in the calibration sample (such as 1000 μg / L) is the corresponding conversion factor, A IS(3,5 / 2,6-DMP)，1 and A IS(3,5-DMP)，1 The peak area of ​​the internal standard quantitation precursor ion in the calibration sample is calculated for the calibration points and conversion factors of the above calibration sample series.

[0118] The calibration samples related to the first conversion factor may be repeatedly measured, and the average value thereof may be taken as k1.

[0119] Similarly, a calibration curve is drawn with the sum of the concentrations of 3,5-dimethylphenol and 2,6-dimethylphenol as the independent variable and the sum of the response values ​​(such as peak area) of the 93.03459 m / z product ions of 3,5-dimethylphenol and 2,6-dimethylphenol divided by the product ion response value (such as peak area) of its associated internal standard as the dependent variable. The concentration value of the co-eluting component calculated by the calibration curve is defined as the second standardized concentration of the co-eluting 3,5-dimethylphenol and 2,6-dimethylphenol, and is expressed as c STD2 express.

[0120] The second conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol is represented by k2 and is calculated according to formula (2):

[0121]

[0122] Among them, A 3,5 / 2,6-DMP，2 A is the quantitative product ion peak area of ​​the co-eluting components 3,5-dimethylphenol and 2,6-dimethylphenol at the calibration point (e.g. 1000 μg / L) of the calibration sample series, 3,5-DMP，2 The quantitative product ion peak area of ​​3,5-dimethylphenol in the calibration sample (such as 1000 μg / L) is the corresponding conversion factor, A IS(3,5 / 2,6-DMP)，2 and A IS(3,5-DMP)，2 The peak area of ​​the corresponding internal standard quantifier ion in the calibration sample is calculated for the calibration points and conversion factors of the above calibration sample series.

[0123] The relevant calibration samples of the second conversion factor can be repeatedly measured and the average value thereof can be taken as k2.

[0124] The concentration of 3,5-dimethylphenol in the sample c 3,5-DMP and the concentration of 2,6-dimethylphenol c 2,6-DMP , and c STD1 、c STD2 The relationship between k1 and k2 can be expressed by formula (3) and formula (4):

[0125]

[0126]

[0127] Solving formula (3) and formula (4) together, we have

[0128]

[0129]

[0130] 5. Calibration range and detection limit

[0131] The linear regression correlation coefficients (R) of 13 phenolic components in the range of 10 to 5000 μg / L 2 ) were all greater than 0.997. The detection limits of the 13 phenolic components were all ≤1.0μg / L. Compared with the standard HJ744-2015 (derivative-GC-MS method) for testing phenolic compounds, the sensitivity of this method was 1 to 2 orders of magnitude higher.

[0132] It should be noted that this embodiment is only a preferred example. The present invention does not limit the type and specification of the chromatographic column and the type of LC-HRMS instrument. Other similar reverse chromatographic columns can be used to implement the present invention according to the technical solution disclosed in the present invention.

[0133] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A high-resolution mass spectrometry characterization method for chromatographic co-elution of dimethylphenol isomers, characterized in that: The method comprises the following steps: According to the petroleum content range in the sample, select the corresponding sample pretreatment process to prepare the sample; In the mobile phase system of methanol-5.0~6.5mmol / L ammonia water, the standard sequence samples, the conversion factor calibration samples and the sample were detected by LC-HRMS, and the primary mass spectrometer full scan (MS 1 ) and selected ion secondary mass spectrometry (MS 2 )data; According to MS 1 data, calculate the first standardized concentrations of coeluting 3,5-dimethylphenol and 2,6-dimethylphenol, and the first conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol by internal standard calibration; and the concentrations of phenol, 2-methylphenol, 3-methylphenol, 4-methylphenol, 2,3-dimethylphenol, 2,4-dimethylphenol, 2,5-dimethylphenol, 3,4-dimethylphenol, 2-ethylphenol, 3-ethylphenol, and 4-ethylphenol; According to MS 2 The data were used to calculate the second standardized concentrations of co-eluted 3,5-dimethylphenol and 2,6-dimethylphenol, and the second conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol by internal standard calibration method; The concentrations of 3,5-dimethylphenol and 2,6-dimethylphenol are calculated based on the first standardized concentration and the second standardized concentration of 3,5-dimethylphenol and 2,6-dimethylphenol; and the first conversion factor and the second conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol.

2. The high-resolution mass spectrometry characterization method for chromatographically co-eluting dimethylphenol isomers according to claim 1, characterized in that: According to the petroleum content range in the sample, the corresponding sample pretreatment process is selected to prepare the sample, which specifically includes: When the petroleum content in the sample does not exceed 50 mg / L, the sample is filtered through a 0.22 μm filter membrane before sampling and testing; When the petroleum content in the sample exceeds 50 mg / L, the sample is alkalized with NaOH and extracted with dichloromethane and n-hexane, and the organic phase is separated by centrifugation. The aqueous phase is filtered through a 0.22 μm filter membrane, and then neutralized with formic acid before sampling and testing.

3. The high-resolution mass spectrometry characterization method for chromatographically co-eluting dimethylphenol isomers according to claim 1 or 2, characterized in that: The elution conditions of the methanol-5.0-6.5 mmol / L ammonia water mobile phase system include: The column flow rate was 0.20 mL / min; Gradient elution was as follows: maintaining zero gradient elution for 1 min, then isocratic elution to increase the volume ratio of methanol to 40% within 30 min, and maintaining the volume ratio of methanol at 40% until all components were eluted; The column temperature is 35-40°C.

4. The high-resolution mass spectrometry characterization method for chromatographically co-eluting dimethylphenol isomers according to claim 3, characterized in that: The elution conditions of the methanol-5.0-6.5 mmol / L ammonia water mobile phase system include: The column flow rate was 0.20 mL / min; Gradient elution: 0-1 min, 20% methanol; 1-31 min, 20%-40% methanol; 31-32 min, 40% methanol; 32-32.1 min, 40%-90% methanol; 32.1-37 min, 90% methanol; 37-37.1 min, 90%-20% methanol; 37.1-42 min, 20% methanol; The column temperature was 40°C.

5. The high-resolution mass spectrometry characterization method for chromatographically co-eluting dimethylphenol isomers according to claim 1, characterized in that: The selected ion secondary mass spectrometry (MS 2 ), the parent ion of 3,5-dimethylphenol-2,4,6-D3 is 124.08472 m / z, and the quantitative daughter ion is 96.05342 m / z; the parent ions of 3,5-dimethylphenol and 2,6-dimethylphenol are 121.06589 m / z, and the quantitative daughter ion is 93.03459 m / z.

6. The high-resolution mass spectrometry characterization method for chromatographically co-eluting dimethylphenol isomers according to claim 1, characterized in that: The calculation formula of the first conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol is: Where k1 is the first conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol, A 3,5 / 2,6-DMP，1 is the quantitative precursor ion peak area of ​​the co-eluting components 3,5-dimethylphenol and 2,6-dimethylphenol in the calibration points of the calibration sample series, A 3,5-DMP，1 The quantitative parent ion peak area of ​​3,5-dimethylphenol in the calibration sample is A IS(3,5 / 2,6-DMP)，1 and A IS(3,5-DMP)，1 The peak areas of the corresponding internal standard quantitation precursor ions in the calibration samples are calculated for the calibration points and conversion factors of the calibration sample series.

7. The high-resolution mass spectrometry characterization method for chromatographically co-eluting dimethylphenol isomers according to claim 1, characterized in that: The calculation formula of the second conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol is: Where k2 is the second conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol, A 3,5 / 2,6-DMP，2 is the quantitative product ion peak area of ​​the co-eluting components 3,5-dimethylphenol and 2,6-dimethylphenol at the calibration points of the calibration sample series, A 3,5-DMP，2 The quantitative product ion peak area of ​​3,5-dimethylphenol in the calibration sample is A IS(3,5 / 2,6-DMP)，2 and A IS(3,5-DMP)，2 The peak areas of the corresponding internal standard quantifier ions in the calibration samples are calculated for the calibration points and conversion factors for the calibration sample series.

8. The high-resolution mass spectrometry characterization method for chromatographically co-eluting dimethylphenol isomers according to claim 1, characterized in that: The calculation formula for the concentration of 3,5-dimethylphenol and 2,6-dimethylphenol is: In the formula, c 2,6-DMP represents the concentration of 2,6-dimethylphenol, c 3,5-DMP represents the concentration of 3,5-dimethylphenol, c STD1 represents the first normalized concentration of coeluting 3,5-dimethylphenol and 2,6-dimethylphenol, c STD2 represents the second standardized concentration of co-eluted 3,5-dimethylphenol and 2,6-dimethylphenol, k1 represents the first conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol, and k2 represents the second conversion factor of 2,6-dimethylphenol to 3,5-dimethylphenol.

9. An application of the high-resolution mass spectrometry characterization method for chromatographically co-eluting dimethylphenol isomers as claimed in any one of claims 1 to 8, characterized in that: The characterization method is applied to the quantitative characterization of 13 C0-C2-alkyl substituted phenol single components in oily wastewater, coal gasification wastewater, oily solid waste, drinking water, surface water and groundwater.

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